Protein Production
Proteins can be recombinantly produced using a vast array of expression systems in a wide variety of cells such as bacteria, yeast, insect cells, and mammalian cells. While the same protein produced in these various systems will generally have the same amino acid sequence, the exact form of the protein and amount produced can differ significantly depending upon which expression system and cell type/organism is used for production.
For many mammalian proteins, expression in bacterial expression systems does not produce functional protein because bacteria and other prokaryotes do not post-translationally modify proteins in the same manner as eukaryotic cells. Such post translational modifications include glycosylation, phosphorylation, and signal peptide cleavage. In order to produce functional versions of these proteins, they may have to be expressed in mammalian cells. However, expression of proteins in mammalian cells can be a time consuming and expensive process.
As an alternative to mammalian cell expression, many proteins may be produced in functional form in insect cells. Production of foreign protein in insect cells is generally considered more cost effective and efficient relative to mammalian cell expression and may be preferred where protein with similar or equivalent biological activity can be produced in insect cells. For example, production of biologically active follicle stimulating hormone and α-galactosidase in insect cells has been described. See U.S. Pat. Nos. 6,183,987; 5,658,567; and 5,179,023. Insect cells which are used for expression of foreign proteins, typically via infection with a recombinant baculovirus, accomplish most of the same post-translational modifications as mammalian cells, including phosphorylation, N- and O- linked glycosylation, acylation, disulphide cross-linking, and oligomeric assembly.
However, post-translational modifications in insect cells are not identical to those that occur in mammalian cells, and these differences are not completely understood. See, e.g., Davidson, D. J. et al., Biochemistry 29: 5584–5590 (1990); Davidson, D. J. et al., Biochemistry 30: 6165–6174 (1991); Jarvis, D. L. et al., Virology 212(2): 500–511 (October 1995); Ogonah, O. W. et al., Nat. Biotechnology 14: 197–202 (1996); Wagner, R. H. et al., J. Virol 70: 4103–4109 (1996); Hsu, T. A. et al., J. Biol. Chem. 272(14): 9062–9070 (April. 1997); Hollister, J. et al., Glycobiology 11: 1–9 (2001); Seo, N. S. et al., Protein Expr. Purif. 22(2):234–41 (2001); Jarvis, D. L. et al., J. Viro. 75(13): 6223–6227 (2001); Kawar, Z. et al., J. Biol. Chem 276(19): 16335–16340 (2001). These differences and their ill-defined nature are generally considered a disadvantage of producing proteins in insect cells. See, e.g. Jarvis, D. L. et al., Curr. Opin. Biotechnology 9(5): 528–533 (October 1998); Marchal, I. et al., Biol. Chem. 382(2): 151–159(2001).
Lysosomal Storage Disorders
Lysosomal storage disorders (LSDs) are a group of genetically inherited disorders that are characterized by a deficiency of one or more specific lysosomal enzymes which causes an accumulation of undigested material (macromolecules) inside the lysosome. This accumulation causes lysosomes to enlarge, leading eventually to cell degeneration. This process results in accumulation of macromolecules in various tissues and organs of the body causing these organs to function less efficiently, resulting in progressive deterioration in physical and/or mental state, and eventually death. A list of LSDs and their associated enzyme deficiency is provided in the following table 1.
TABLE 1Lysosomal Storage DisordersDisease Name/SynonymsEnzyme DeficiencySupporting ReferencePompe DiseaseAcid α-1,4 and 1,6 glucosidaseGenbank: X55079Type II Glycogen Storage(Acid maltase)DiseaseGM1 Gangliodsidosisβ-GalactosidaseGenbank: M34424OMIM: 230500OMIM: 230650OMIM: 230600Tay-Sachs Diseaseβ-Hexosaminidase AGenbank: AH003579GM2 GangliosidosisGM2 Gangliosidosis: ABGM2 Activator ProteinGenbank: L01439VariantSandhoff Diseaseβ-Hexosamindase A & BGenbank: AH002718GM2 GangliosidosisFabry Diseaseα-Galactosidase AGenbank: U78027TrihexosylceramidosisGaucher DiseaseGlucocerebrosidaseGenbank: M19285Glucosylceramide Lipidosisβ-glucosidaseKrabbe DiseaseGalactosylcebrosidaseGenbank: D25283Galactosylceramide Lipidosisβ-GalactosidaseGlobid-Cell LeukodystrophyNiemann-Pick, Types A and BAcid SphingomyelinaseGenbank: M81780Sphingomyelin-CholesterolLipidosisNiemann-Pick, Type CNPC1Genbank: AF002020Transport of Cholesterol topost-lysosomal destinationsNiemann-Pick, Type DNPC1Genbank: AF002020Transport of Cholesterol topost-lysosomal destinationsFarber DiseaseAcid CeramidaseGenbank: U70063Farber LipogranulomatosisCeramidase DeficiencyWolman DiseaseAcid LipaseGenbank: M74775Cholesterol Ester StorageAcid LipaseGenbank: M74775DiseaseHurler Syndromeα-L-IduronidaseGenbank: M74715Mucopolysaccharidosis I(MPS IH/S)Scheie Syndromeα-L-IduronidaseGenbank: M74715Mucopolysaccharidosis I(MPS 1 S)Hurler-Scheieα-L-IduronidaseGenbank: M74715Mucopolysaccharidosis I(MPS IH/S)Hunter SyndromeIduronate 2-SulfataseGenbank: AH000819Mucopolysaccharidosis II (MPSGenbank: M58342II)Sanfilippo A (MPS IIIA)α-N-AcetylglucosaminidaseGenbank: U43572Sanfilippo B (MPS IIIB)α-N-AcetylglucosaminidaseGenbank: U43572Sanfilippo C (MPS IIIC)Acetyl-CoA-GlucosaminideOMIM: 252930AcetyltransferaseSanfilippo D (MPS IIID)N-Acetylglucosamine-6-Genbank: Z12173SulfataseMorquio A (MPS IVA)N-Acetylgalactosamine-6-Genbank: AH006681Sulfate SulfataseMorquio B (MPS IVB)β-GalactosidaseGenbank: M34424OMIM: 253010Maroteaux-Lamy (MPS VI)Arylsulfatase BGenbank: M32373Sly Syndrome (MPS VII)β-GlucuronidaseGenbank: M15182Metachromatic LeukodystrophyArylsulfatase A (cerebrosideGenbank: U62317sulfatase)Genbank: X52151Multiple Sulfatase DeficiencyArylsulfatase A, B and COMIM: 272200Sialidosis (Mucolipidosis I)α-Neuraminidase (glycoproteinOMIM: 256550neuraminidase)Bonten, E. J. et al., J.Biol. Chem. 275: 37657(2000)Genbank: AF040958I-Cell DiseaseUDP GlcNAc: lysosomal-OMIM: 252500Mucolipidosis II (ML-II)enzyme N-Acetylglucosamine-1-phosphotransferasePseudo-Hurler PolydystrophyUDP GlcNAc: lysosomal-OMIM: 252500Mucolipidosis III (ML-III)enzyme N-Acetylglucosamine-1-phosphotransferaseMucolipidosis IV (ML-IV)Mucolipin-1Genbank: AF287269α-Mannosidosisα-MannosidaseGenbank: AH006687β-Mannosidosisβ-MannosidaseGenbank: U60337Fucosidosisα-L-FucosidaseGenbank: M29877AspartylglucosaminuriaN-Aspartyl-β-GlucosaminidaseGenbank: X55330Galactosialidosis (GoldbergProtective Protein/Cathepsin AGenbank: M22960Syndrome)(PPCA), neuraminidase, andOMIM: 256540β-GalactosidaseRudenko, G. et al.,Structure, 3, 1249(1995); Rudenko, G. etal., Proc. Natl. Acad.Sci. USA 95: 621(1998); Bonten, E. J. etal., J. Biol. Chem. 270:26441 (1995)Schindler Diseaseα-N-Acetyl-GalactosaminidaseGenbank: M62783CystinosisCystine Transport ProteinGenbank: AJ222967Salla DiseaseSialinGenbank: AJ387747Infantile Sialic Acid StorageSialinGenbank: AJ387747DisorderBatten Disease (JuvenileUnknownGenbank: U32680Neuronal Ceroid LipofuscinosisInfantile Neuronal CeroidPalmitoyl-Protein ThioesteraseGenbank: U44772LipofuscinosisProsaposinSaposins A, B, C or DGenbank: J03077
A number of LSDs have been treated using enzyme replacement therapy and several clinical trials are ongoing in this area. For example, α-Galactosidase A has been used to treat Fabry disease and glucocerebrosidase has been used to treat Gaucher Disease (sold as Cerezyme® by Genzyme Corp.). Additional examples can be found in the following references:
Pastores G M, Thadhani R., “Enzyme-replacement therapy for Anderson-Fabry disease”, Lancet 358(9282):601–3 (August 2001).
Lin L, Lobel P., “Production and characterization of recombinant human CLN2 protein for enzyme-replacement therapy in late infantile neuronal ceroid lipofuiscinosis”, Biochem J. 357(Pt 1):49–55. (July 2001).
Schiffmann R. et al., “Enzyme replacement therapy in Fabry Disease: a randomized controlled trial” JAMA. 285(21):2743–9 (June 2001)
Desnick R J., “Enzyme replacement and beyond” J Inherit Metab Dis. 24(2):251–65 (April 2001).
Wraith, J. E., “Enzyme replacement therapy in mucopolysaccharidosis type I: progress and emerging difficulties” J Inherit Metab Dis. 24(2):245–50 (April 2001).
Berg, T. et al., “Purification and characterization of recombinant human lysosomal alpha-mannosidase” Mol Genet Metab. 73(1):18–29 (May 2001).
Sly, W. S. et al., “Active site mutant transgene confers tolerance to human beta-glucuronidase without affecting the phenotype of MPS VII mice” Proc Natl Acad Sci U S A. 98(5):2205–10 (February 2001).
Aoki, M. et al., “Improvement of neurological symptoms by enzyme replacement therapy for Gaucher disease type IIIb”, Eur. J. Pediatr. 160(1):63–4 (January 2001).
Ida, H. et al., “Effects of enzyme replacement therapy in thirteen Japanese paediatric patients with Gaucher disease”, Eur. J Pediatr. 160(1):21–5 (January 2001)
Eng, C. M. et al., “A phase ½ clinical trial of enzyme replacement in fabry disease: pharmacokinetic, substrate clearance, and safety studies”, Am J Hum Genet. 68(3):711–22 (March 2001).
Kakkis, E. D. et al., “Enzyme-replacement therapy in mucopolysaccharidosis I”, N. Engl. J. Med. 18;344(3):182–8 (January 2001).
Ioannou, Y. A. et al., “Fabry disease: preclinical studies demonstrate the effectiveness of alpha-galactosidase A replacement in enzyme-deficient mice”, Am. J. Hum. Genet. 68(1):14–25 (January 2001).
Turner, C. T. et al., “Enzyme replacement therapy in mucopolysaccharidosis I: altered distribution and targeting of alpha-L-iduronidase in immunized rats”, Mol. Genet. Metab. 69(4):277–85 (April 2000).
Byers, S. et al., “Delayed growth and puberty in patients with Gaucher disease type 1: natural history and effect of splenectomy and/or enzyme replacement therapy” Isr. Med. Assoc. J. 2(2):158–63 (February 2000).
All of the currently approved treatments and clinical trials in this area as exemplified above utilize enzymes produced in mammalian cells. Some of these enzymes associated with LSDs have also been reported to have been expressed in insect cells in the references cited below, but none of these references report the use of insect-produced proteins in humans.
Chen, Y. et al., “Purification and characterization of human alpha-galactosidase A expressed in insect cells using a baculovirus vector”, Protein Expr. Purif. 20(2):228–36 (November 2000).
Tilkom, A. C. et al., “High-level baculoviral expression of lysosomal acid lipase”, Methods Mol Biol. 109:177–85 (1999).
Steed, P. M. et al., “Characterization of recombinant human cathepsin B expressed at high levels in baculovirus”, Protein Sci. 7(9):2033–7 (September 1998).
Bromme, D., McGrath, M. E., “High level expression and crystallization of recombinant human cathepsin S”, Protein Sci. 5(4):789–91 (April 1996).
Aeed, P. A., Elhammer, A. P., “Glycosylation of recombinant prorenin in insect cells: the insect cell line Sf9 does not express the mannose 6-phosphate recognition signal”, Biochemistry 33(29):8793–7 (July 1994).
Coppola, G. et al., “Characterization of glycosylated and catalytically active recombinant human alpha-galactosidase A using a baculovirus vector”, Gene 144(2): 197–203 (July 1994).
Boose, J. A. et al., “Synthesis of a human lysosomal enzyme, beta-hexosaminidase B, using the baculovirus expression system”, Protein Expr. Purif 1(2): 111–20 (November 1990).
Martin, B. M. et al., “Glycosylation and processing of high levels of active human glucocerebrosidase in invertebrate cells using a baculovirus expression vector”, DNA 7(2):99–106 (March 1988).
Wu, J. Y. et al., “Expression of catalytically active human multifunctional glycogen-debranching enzyme and lysosomal acid alpha-glucosidase in insect cells”, Biochem. Mol. Biol. Int. 39(4):755–64 (July 1996).